Lentiviral vectors, including those derived from HIV-1, hold great promise for gene therapy. However, the possibility of generating replication competent retrovirus (RCR) through genetic recombination raises serious concerns for safety. Given the limitations of available animal models to evaluate these vectors in vivo, safety will ultimately be determined in human hosts. Therefore, it is imperative that the design of the vector itself ensures the maximal level of safety attainable. Using a highly sensitive assay that we developed to specifically select for genetic recombinants, we have demonstrated that recombination occurs between the packaging and vector components of the 3rd generation and SIN lentiviral vector systems in transduced cells. Importantly, we hay shown that these envelope-minus recombinants can express functional gag/gag-pol, which is capable c mobilizing retroviral DNA when exogenous envelope is provided in trans. Based on our earlier findings that full functional reverse transcriptase (RT) and integrase (IN) can be incorporated into HIV- 1 particles in trans, independently of Gag-Pol, we have developed a vector ("trans-lentiviral" vector) that prevents the generation a recombinants that contain RI-IN (gag-pol). Since functional gag-pol is absolutely required for the emergence or any type of RCR and for retroviral DNA mobilization, the trans-lentiviral vector improves safety in two important ways: First, an additional recombination events involving RT-IN is necessary to generate recombinants containing a functional gag-pol structure, and second, the trans-lentiviral vector design itself makes it possible to monitor vector stocks in vitro for the regeneration of a functional gag-pol structure. Our central hypothesis is that the trans-lentiviral vector design will ensure the greatest level of safety that is predictable while retaining the ability to produce high titer vector stocks capable of efficient transduction of nondividing cells. To test our hypothesis, we propose to: (1) Construct gag-pro packaging plasmids containing minimal RT and IN coding sequences; (2 Genetically modify the trans-RT-IN expression construct; (3) Analyze the trans-lentiviral packaging an trans-RT-IN constructs for genetic recombination; (4) Establish an inducible stable packaging cell line capable c producing high titer trans lentiviral vector; (5) Evaluate the trans-lentiviral vector for transduction o hematopoietic stem cells.